The prior art has employed various devices to measure the fluorescence of materials. Fluorometers illuminate materials with selected wavelengths of light and measure the fluorescence emitted at specific wavelengths which are longer than those used to illuminate the material. Conventional laboratory spectrofluorometers employ rectangular quartz sample chambers wherein the excitation source illuminates the sample through one side of the chamber and the emitted fluorescence is measured through another side of the chamber. The emitted light may be measured through the window at 90.degree. or 180.degree. with respect to the window used to illuminate the sample. Instruments of this design are generally used to analyze discreet samples off-line with respect to the process. Flow-through chambers are also available for a limited number of on-line applications.
Instruments with sample chambers are unsuited for most on-line applications. There are many practical difficulties with supplying samples to flow cells which are representative of the contents of a larger vessel. In highly light-absorbant samples, only the layer of sample immediately adjacent to the window through which the illuminating light passes will fluoresce. The emitted light cannot reach the window on which the detector is focused. In dilute samples where only a portion of the illuminated light is absorbed by the sample, greater sensitivity could be achieved if the sample chamber were enlarged so that more fluorophores were present in the field of view for the detector. This suggests that sample chamber sizes should vary with sample characteristics to maximize sensitivity. Alternatively, samples could be diluted to a specific fluorescence value. Both procedures are obviously impractical for on-line applications. In many cases, however, these instruments are not suitable for on-line applications because of poor sensitivity to highly light-absorbent samples and difficulties in supplying samples to a flow cell which are representative of the contents of a larger reactor.
Other fluorometer designs employ surface detectors which use a single transparent window to illuminate the material and observe the emitted fluorescence. The surface detector principle is reported to have greater sensitivity with highly light-absorbent samples than instruments that use the rectangular sample chamber. (Photoluminescence of Solutions, With Applications to Photochemistry and Analytical Chemistry, C. A. Parker, 1968, pp. 226-229). The surface detector principle also permits improved on-line process capabilities. For example, one instrument has been described which was attached to the side of a glass fermentor to obtain on-line fluorescence measurements of the culture within the fermentor. (Fluorimetric Technique for Monitoring Changes in the Level of Reduced Nicotinamide Nucleotides in Continuous Cultures of Microorganisms, D. E. F. Harrison and Britton Chance, 1969, pp. 446-450). This design used a lamp source and detector aimed at a common spot on the surface of the window adjacent to the sample and at 60.degree. to one another. The practical limitations to this design are many. The application is dependent on a transparent vessel or one equipped with a window. With a fixed angle between them, this requires that the distance between the sample interface and the instrument be fixed at an optimal value. Since this distance is partly a function of the vessel window assembly, the instrument must be custom designed to each vessel type. Mixing is poor at any vessel surface so that the material nearest the fluorometer may not be representative of the vessel contents. The long term stability of this instrument was low. (Applied and Environmental Microbiology, Estimation of Fermentation Biomass Concentration by Measuring Culture Fluorescence, D. W. Zabriskie and A. E. Humphrey, 1978, pp. 337-343). It also makes inefficient use of the illuminating light at low concentrations of fluorophore. In this case, the excitation light may penetrate several feet into the tank. Since the detector is looking in a direction 60.degree. from the path of illumination, the amount of observable fluorescence is low.
A probe is described which uses a complicated system of lenses to collimate the illuminating light and emitted fluorescence, and a dichroic mirror to separate the paths of the illuminating lights and emitted fluorescence to form an angle of 90.degree. with respect to one another. (European Journal of Applied Microbiology and Biotechnology, On-Line Measurements of Culture Fluorescence: Method and Application, W. Beyeler, A. Einsele, and A. Fiechter, pp. 10-14, 1981). This system makes poor use of light since the dichroic mirror is a relatively inefficient device and light losses through refraction are large owing to the number of surfaces associated with the lenses, filters, and dichroic mirror. These factors made it necessary to cool the probe using an external source of cooling water in order to increase instrument sensitivity since the detector performance is inversely related to temperature. A practical lower temperature limit of 15.degree. C. was realized however, since lower temperatures lead to water condensation from the atmosphere inside the probe. This probe is not well suited to industrial use. The need to separate the paths for the illuminating light and emitted fluorescence resulted in an unsymmetrical and bulky configuration difficult to seal from the operating environment. The lenses and their proper alignment are fragile. The lenses must be fabricated from non-fluorescent materials and are expensive, especially when manufactured from quartz for UV applications. Since the performance of the dichroic mirror and lens system must be optimized for a specific combination of illuminating light and emitted fluorescence wavelengths, probe modifications are required when it is desired to change the wavelength combination. Moreover, because of the inherent fixed refraction characteristics of the lenses, and because the illuminating light has a different wavelength from that of the emitted fluorescence, the apparatus provides a different field of illumination for the illuminating light than the field of view of the detector measuring the emitted fluorescence, which is a serious disadvantage, as will further become apparent.
The term "field of view" for an optical detector as used herein refers to the region within a sample which is observable by the detector. The term "field of illumination" for an illuminating light is defined herein as the region within a sample which is illuminated by the excitation light.
Another system uses a bifurcated fiber optic bundle for this purpose. (Science, Vol. 217, Aug. 6, 1982, Intracellular Oxidation-Reduction State Measured in situ by a Multichannel Fiber-Optic Surface Fluorometer, Avraham Mayevsky and Britton Chance, pp. 537-540). Here too inefficiencies in the fiber optics require the use of a light source and detector unsuitable for incorporation into a probe of practical size. These inefficiencies relate to the loss of UV and visible light during transmission through the fibers and a disadvantageous difference between the field of illumination established by the fibers transmitting the illuminating light and the field of view of the detector established by the fibers transmitting the emitted fluorescence. This becomes especially significant in highly light absorbent samples where the penetration of light into the sample is limited to a very short distance.
Although this invention can be used with a wide range of materials, it is principally intended for the real-time determination by their fluorescence of the characteristics of living biological materials directly in process. The application of fluorescence measurements in biological systems is well known in the art. (Fluorescence Assay in Biology and Medicine, Volume 1, Sidney Undenfriend, 1962, pp. IX-XII). For example, living cells emit visible fluorescent light at 460 nm when illuminated with ultraviolet light at 340 nm. Most of this fluorescence is due to the intracellular accumulations of the reduced forms of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, (NAD(P)H), important energy storage molecules common to living matter. This principle has been used to study metabolism in intact tissues including brain, liver, kidney, and testis tissue. (U.S. Pat. No. 3,313,290, Britton Chance and Victor A. Legallais and Science, Vol. 217, Aug. 6, 1982, Intracellular Oxidation-Reduction State Measured in Situ by a Multichannel Fiber-Optic Fluorometer, Avraham Mayevsky and Britton Chance, pp 537-540). Suspensions of yeast, bacteria and fungi have also been studied with the aid of fluorescence. (Applied and Environmental Microbiology, Estimation of Fermentation Biomass Concentration by Measuring Culture Fluorescence, Feb. 1978, pp. 337-343, D. W. Zabriskie and A. E. Humphrey).